Sequencing of the cloned plasmid - (Jul/22/2013 )

Hi all,
I cloned a 3.2kbp of fragment into pGL3-basic vector and then sent to a company for sequencing with RV3 and GL2 primers.
The sequencing results with RV3 was good, but with GL2 had lots of noise and I could perform alignment.
Do you know what could cause that problem?
Thank you very much for helping me out.
Best.

-DrLeo-

It could be that you have two different forms of the insert in the plasmid, but it is more likely that you have contaminated the GL2 primer.

-bob1-

I would restreak for single colonies, then prepare plasmid again. Also, you could choose another primer and synthesize it, just to make sure it was not a primer design issue. Is your plasmid the expected size? I've seen double-sized plasmids with one correct and one incorrect insert.

-phage434-

Dear bob1 and phage434,
Thanks for your suggestion.
However, if I got 2 different plasmids in one colony, or 2 inserts in one plasmid, the noise should happen with both GL2 and RV3 primers, but it only happened with GL2.
Moreover, I used gel electrophoresis with non-cut and cut plasmid with specific RE to confirm the insert, the bands were totally right in size of insert and plasmid.
So, I think the problem should be due to the contaminated of the GL2 primer...
Do you think so?
Thanks again!

-DrLeo-

DrLeo on Tue Jul 23 01:27:00 2013 said:

However, if I got 2 different plasmids in one colony, or 2 inserts in one plasmid, the noise should happen with both GL2 and RV3 primers,

Not necessarily, as you are sequencing a big insert, the noise could quite easily be seen only on one end if you had more than one insert in the same plasmid. Two different inserts would probably give you noise at both ends, but not if they shared an identical start. Splice variants would be one form of a gene that could give this result.

Cutting out the band won't tell you if you have back to back insertions as they would be digested between the two inserts.